The marine yeast Debaryomyces hansenii is known to tolerate salinities ranging from 0 to 24%. As a first step toward the molecular analysis of halotolerance in this organism, we report the isolation of an autonomously replicating sequence (ARS) and its use in the construction of a shuttle vector. The ARS from D. hansenii (ARSD) is 0.4 kbp long, and the function rests in 0.13 kbp of the sequence. Sequence analysis of ARSD shows strong homology to ARS from other organisms, including a 12-bp consensus sequence common to all ARS functional in Saccharomyces cerevisiae.